(1) Molecular cloning and analysis of mutants phenotypically similar to mig-17

To under stand the function of MIG-17 in DTC migration, we are isolating and analyzing mutants having a meandering DTC phenotype similar to the mig-17 mutants. In mig-23, mig-29 and mig-30 mutants, MIG-17 secreted from the muscle cells failed to localize to the gonad. mig-23 encodes Golgi NDPase required for protein glycosylation, and mig-29 (cogc-3) and mig-30 (cogc-1) encode two of the eight subunits of the conserved oligomeric Golgi (COG) complex required for vesicle transport (Nishiwaki et al., Nat. Cell Biol. 6, 31-37, 2004; Kubota et al., Development 133, 263-273, 2006). COGC-1 and COGC-3 normally stabilize MIG-23 in the Golgi and contribute for proper glycosylation of MIG-17, which is requisite for MIG-17 localization to the gonad. We continue to analyze mutants with meandering DTC phenotypes.

(2) Molecular cloning and analysis of the mig-17 suppressors

In order to identify genes interacting with mig-17, we are isolating suppressors of mig-17 mutations. Two different mig-17 mutations are used: mig-17(k174), a null mutation and mig-17(k167), a missense mutation in the metalloprotease domain. Both of these mutations show similar phenotypes, but k174 mutation is stronger than k167, suggesting that k167 is a partial loss-of-function mutation. We expect to identify mutations affecting downstream events of MIG-17 or those that bypass the requirement of MIG-17 from k174 suppressors. We also expect to isolate mutations in genes that negatively regulate MIG-17 activity from k167 suppressors. For example, we might be able to identify mutations in an inhibitor protein of the MIG-17 activity. 11 suppressor mutations isolated from k174 were grouped into at least 6 different loci. We will clone and molecularly analyze the suppressor genes.

(3) Analysis of genetic interactions between mig-17 or suppressor genes and genes for      extracellular matrix proteins

Although C. elegans is thought to be phyletically separated from vertebrates by 800 million years, it has a well conserved repertoire of extracellular matrix (ECM) proteins. These include nidogen, proteoglycans (perlecan, syndecan, agrin, glypican, typeXVIII collagen, NG2, etc.), laminin, type IV collagen, SPARK, fibulin and fibrillin (Hutter et al., Science 287, 989-994, 2000). As C.elegans is essentially devoid of an interstitial matrix, most of these proteins are likely to be components of basement membranes. Importantly, C. elegans often has fewer isoform-encoding genes for ECM proteins compared to vertebrates. For example, C. elegans has only one fibulin gene (fbl-1), whereas mammals have five fibulin genes. It is therefore easier to genetically analyze the functions of these molecules in C. elegans. We will examine whether there are any genetic interactions between mig-17 or suppressor genes and the genes for other ECM proteins.

(4) Biochemical analysis of interacting proteins for MIG-17 and suppressor gene products

Although the genetic screening of interacting molecules is a powerful approach available in C. elegans, it may not be successful in identifying such molecules when they involve functionally redundant genes or mutations which result in lethality. To identify molecules which physically interact with MIG-17 or suppressor proteins, we will carry out coimmunoprecipitation experiments, affinity screening with a column fixed with MIG-17 or suppressor proteins produced by the baculovirus expression system and screening with the yeast two-hybrid system. Once the interacting proteins are found, it will be possible to disrupt the genes for such proteins for functional analysis by a reverse genetics approach.

(5) Development of an in vitro culture system of C. elegans gonad

C. elegans gonad primordium is formed in late embryonic development. The primordium first adheres to the ventral body wall muscle at the first larval (L1) stage, and the DTCs generated in late L1 stage begin to migrate along the ventral muscle at the L2 stage. In dig-1 mutants, the gonad primordium is often missattached to the dorsal body wall muscle or lateral hypodermis (Thomas et al., Cell 62, 1041-1052, 1990; K. Nishiwaki, unpublished). When this occurs functional gonads are still formed, although the morphology of the gonad arms becomes abnormal. It seems that gonad development is not strongly dependent on its position in the body, raising the possibility that the isolated gonad primordium might be able to develop to a certain stage in vitro by devising an appropriate culture medium. We will try to develop in vitro culture system of the C. elegans gonad.